Insecticidal Plant Cyclotide with Activity Against Homopteran Insects

ABSTRACT

The present invention relates to isolated nucleic acids encoding plant cyclotides. The invention also relates to the construction of a chimeric gene encoding all or a portion of the plant cyclotides, in sense or antisense orientation, wherein expression of the chimeric gene results in the production of altered levels of plant cyclotides in a transformed host cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/616,190, filed Oct. 5, 2004 and U.S. Ser. No. 11/236,922, filedSep. 28, 2005, the contents of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

Embodiments of the present invention relate to naturally-occurring andrecombinant nucleic acids encoding cyclotides characterized by activityagainst plant pathogens. Compositions and methods of certain embodimentsof the invention utilize the disclosed nucleic acids, and their encodedpolypeptides to control plant pathogens.

BACKGROUND OF THE INVENTION

Plant pathogens are responsible for significant annual crop yieldlosses. One strategy for the control of plant pathogens is the use ofresistant cultivars selected for, or developed by, plant breeders forthis purpose. However, novel mechanisms for pathogen resistance can beimplemented more quickly by molecular methods of crop protection than bytraditional breeding methods. Accordingly, molecular methods are neededto supplement traditional breeding methods to protect plants frompathogen attack.

Plants rely heavily on a chemical and biological armory for theirdefense from a variety of pests and pathogens. Small cysteine-richproteins that have been implicated in host defense and isolated fromplant sources include defensins, thionins, and small antimicrobialproteins (AMP's). Cyclotides, also cysteine-rich molecules, haverecently been recognized and characterized as being involved in hostdefense (Craik et al. (1999), J. Mol. Biol. 294: 1327-1336; Craik et al.(2000), Toxicon 39: 43-60). Cyclotide polypeptides are encoded by genesequences, are produced as linear precursors, are cysteine-rich, and arecapable of being cyclized via a peptide bond. Cyclotides display adiverse range of biological activities such as antibacterial activity,antifungal activity, anti-HIV activity, and uterotonic activity (Craik(2001), Toxicon 39: 1809-1813). Cyclotides have additionally been shownto possess insecticidal activity (Jennings et al. (2001) Proc. Natl.Acad. Sci. U.S.A. 98:10614-10619). Cyclized cyclotides differ fromclassical proteins in that they have no free N- or C-terminus due totheir amide-circularized backbone.

Cyclotide polypeptides are derived from longer precursor proteins andthus both cleavage and cyclization steps are involved in the productionof the cyclic backbone. The cyclic backbone of the cyclotide moleculetypically ranges in size from 29 to 37 amino acid residues and has threedisulfide bonds that form a cystine knot motif where two disulfide bondsand their connecting backbone strands form a ring that is threaded bythe third disulfide bond. The mechanism(s) inherent to backbonecyclization is currently not known. One possibility is enzymatic orchemical involvement in both the backbone cleavage of the mature domainand the subsequent cyclization. The combined features of the cycliccystine knot produce a unique protein fold that is topologically complexand has exceptional chemical and biological stability.

The majority of the plant cyclotides have been isolated from Rubiaceaeand Violaceae plants (Gustafson et al. (1994), J. Nat. Prod. 116:9337-9338; Gustafson et al. (2000), J. Nat. Prod. 63: 176-178; Witherupet al. (1994), J. Nat. Prod. 57: 1619-1625; Saether et al. (1995),Biochemistry 34, 4147-4158; Bokesch et al. (2001), J. Nat. Prod. 64:249-250; Schöpke et al. (1993), Sci. Pharm. 61: 145-153; Claeson et al.(1998), J. Nat. Prod. 61: 77-81; Göransson et al. (1999), J. Nat. Prod.62: 283-286; Hallock et al. (2000), J. Org. Chem. 65: 124-128;Broussalis et al. (2001), Phytochemistry 58: 47-51). Recently, twomembers of a new sub-class of the cyclotide family have been discoveredin Curcurbitaceae (Hernandez et al. (2000), Biochemistry 39: 5722-5730.;Felizmenio-Quimio et al. (2001), J. Biol. Chem. 276: 22875-22882; Heitzet al. (2001), Biochemistry 40: 7973-7983; Trabi and Craik, (2002),Trends in Biochem. Sci. 27: 132-138).

Cyclotides may be used in transgenic plants in order to produce plantswith increased resistance to pathogens such as fungi, viruses, bacteria,nematodes, and insects. Thus, embodiments of the present invention solveneeds for the enhancement of a plant's defensive response via amolecularly based mechanism which can be quickly incorporated intocommercial crops.

SUMMARY OF THE INVENTION

Compositions and methods relating to pathogen resistance are provided.

Embodiments of the invention include a cyclotide sequence which findsuse in enhancing the plant pathogen defense system. Further embodimentsinclude compositions and methods which can be used for enhancing plantresistance to Homopteran insect pests. The method involves stablytransforming a plant with a nucleotide sequence capable of modulatingthe plant pathogen defense system operably linked with a promotercapable of driving expression of a gene in a plant cell.

Transformed plants, plant cells, and seeds, as well as methods formaking such plants, plant cells, and seeds, are additionally provided.It is recognized that a variety of promoters will be useful in thevarious embodiments of the invention, the choice of which will depend inpart upon the desired level of expression of the disclosed genes. It isrecognized that the levels of expression can be controlled to modulatethe levels of expression in the plant cell.

Embodiments of the invention are directed to a cyclizable molecule andits linear precursor; cyclic peptides, polypeptides or proteins; andadditionally includes the linear forms of non-cyclic structuralhomologues of the cyclic peptides, polypeptides and proteins. Alsoincluded are derivative forms of the cyclized molecule and their linearprecursors encoded by the subject nucleic acid molecules. The cyclic andlinear peptides, polypeptides or proteins may be naturally occurring ormay be modified by the insertion or substitution of heterologous aminoacid sequences.

One embodiment of the invention provides an isolated nucleic acidmolecule comprising a sequence of nucleotides, which sequence ofnucleotides encodes an amino acid sequence or a derivative form thereofcapable of being cyclized within a cell or a membrane of a cell to forma cyclic backbone wherein the cyclic backbone comprises sufficientdisulfide bonds to confer a stabilized folded structure on the threedimensional structure of the backbone. The amino acid sequence may alsobe cyclizable in an in vitro system comprising, for example, cyclizingenzymes or a chemical means for cyclization.

Embodiments of the invention also extend to the peptide, polypeptide orprotein sequences which are capable of cyclizing in the absence of anyother exogenous factor and more specifically capable of circularizingthrough a catalytic process being an inherent activity of the peptides,polypeptides or proteins.

Embodiments of the invention comprise a peptide sequence that can beprocessed from a larger polypeptide sequence, more specifically, apeptide sequence which can be cleaved and cyclized. Such embodimentsfurther extend to linear forms and precursor forms of the peptide,polypeptide or protein which may also have activity or other utilities.Other embodiments extend to engineering crop plants with the sequencesof the invention in order to produce plants that are resistant topathogens.

Embodiments of the invention concern an isolated polynucleotidecomprising a nucleotide sequence set forth in SEQ ID NOs: 1, 4 or 5; anucleotide sequence that encodes a polypeptide having the amino acidsequence set forth in SEQ ID NOs: 2, 3, or 6, a nucleotide sequencecharacterized by at least 85% sequence identity to the nucleotidesequences set forth in SEQ ID NOs: 1, 4 and 5; a nucleotide sequencecharacterized by at least 90% sequence identity to the nucleotidesequences set forth in SEQ ID NOs: 1, 4 and 5; a nucleotide sequencecharacterized by at least 95% sequence identity to the nucleotidesequences set forth in SEQ ID NOs: 1, 4 and 5; and a nucleotide sequencethat comprises the complement of any one of the above. A furtherembodiment is the complement of the nucleotide sequences disclosedherein.

Embodiments of the invention also relate to a chimeric gene comprisingan isolated polynucleotide of the present invention operably linked tosuitable regulatory sequences.

A further embodiment of the invention concerns an isolated host cellcomprising a chimeric gene or an isolated polynucleotide. The host cellmay be eukaryotic, such as a yeast or a plant cell, or prokaryotic, suchas a bacterial cell. Another embodiment relates to a virus, such as abaculovirus, comprising an isolated polynucleotide or a chimeric gene.

Another embodiment of the invention provides a process for producing anisolated host cell comprising a chimeric gene or an isolatedpolynucleotide, the process comprising either transforming ortransfecting an isolated compatible host cell with a chimeric gene orisolated polynucleotide.

An embodiment of the invention also provides an isolated polypeptideselected from the group consisting of: a polypeptide comprising an aminoacid sequence set forth in SEQ ID NOs: 2, 3 or 6; a polypeptidecharacterized by at least 90% identity to SEQ ID NOs: 2, 3 or 6; apolypeptide characterized by at least 95% identity to SEQ ID NOs: 2, 3or 6; a polypeptide characterized by at least 97% identity to SEQ IDNOs: 2, 3 or 6; a polypeptide characterized by at least 98% identity toSEQ ID NOs: 2, 3 or 6; and a polypeptide characterized by at least 99%identity to SEQ ID NOs: 2, 3 or 6. The polypeptides are useful inprotecting plants from various Homopteran insect pests including, butnot limited to, corn plant hopper (Peregrinus maidis) and soybean aphid(Aphis glycines).

An embodiment additionally provides a method for impacting a plantHomopteran insect comprising introducing into a plant or cell thereof atleast one nucleotide construct comprising a coding sequence operablylinked to a promoter that drives expression of a plant cyclotidepolypeptide in plant cells, wherein said nucleotide sequence is selectedfrom the group consisting of: a nucleotide sequence set forth in SEQ IDNOs: 1, 4 or 5; a nucleotide sequence that encodes a polypeptide havingthe amino acid sequence set forth in SEQ ID NOs: 2, 3 or 6; a nucleotidesequence characterized by at least 85% sequence identity to thenucleotide sequences set forth in SEQ ID NOs: 1, 4 or 5; a nucleotidesequence characterized by at least 90% sequence identity to thenucleotide sequences set forth in SEQ ID NOs: 1, 4 or 5; a nucleotidesequence characterized by at least 95% sequence identity to thenucleotide sequences set forth in SEQ ID NOs: 1, 4 or 5; and anucleotide sequence that comprises the complement of any one of theabove.

Expression cassettes and stably transformed plants are also provided byembodiments of the invention. Other embodiments provide nucleic acidsand fragments and variants thereof which encode polypeptides or maturepolypeptides that possess activity against plant Homopteran pests.

In a particular embodiment, a transformed plant of the invention can beproduced using a nucleic acid that has been optimized for increasedexpression in a host plant. For example, the cyclotide polypeptides canbe back-translated to produce nucleic acids comprising codons optimizedfor expression in a particular host, for example a crop plant such as asoybean plant or a maize plant. Some embodiments provide transgenicplants expressing polypeptides that find use in methods for impactingplant Homopteran insect pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the HPLC profile of the crude extract from the Viola spp.showing the absorbance measured at 214 nm. The peak corresponding to thecyclotide (SEQ ID NO:3) is labeled “E5.” The mass was observed as 3158.3Da, which agrees well with the theoretical mass of 3159.84 Da, wellwithin the error limit.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention are drawn to compositions and methods forimpacting Homopteran insect pests, particularly plant pests. Morespecifically, the isolated nucleic acids of the embodiments of theinvention, and fragments and variants thereof, comprise nucleotidesequences that encode pesticidal polypeptides (e.g., proteins). Thedisclosed pesticidal proteins are biologically active (e.g., pesticidal)against insect pests such as insect pests of the order Homoptera. Insectpests of interest include, but are not limited to: corn plant hopper(Peregrinus maidis) and soybean aphid (Aphis glycines).

Other embodiments of the invention include compositions comprisingisolated nucleic acids, and fragments and variants thereof that encodepesticidal polypeptides, expression cassettes comprising nucleotidesequences of embodiments of the invention, isolated pesticidal proteins,and pesticidal compositions. Embodiments of the invention furtherprovide plants and microorganisms transformed with these novel nucleicacids, and methods involving the use of such nucleic acids, pesticidalcompositions, transformed organisms, and products thereof in impactinginsect pests.

The nucleic acids and nucleotide sequences described herein may be usedto transform any organism to produce the encoded pesticidal proteins.Methods are provided that involve the use of such transformed organismsto impact or control plant pests. The nucleic acids and nucleotidesequences may also be used to transform organelles such as chloroplasts(McBride et al. (1995) Biotechnology 13:362-365; Kota et al. (1999)Proc. Natl. Acad. Sci. USA 96: 1840-1845).

Embodiments of the invention further provide fragments and variants ofthe naturally occurring coding sequences that also encode biologicallyactive (e.g., pesticidal) polypeptides. These nucleotide sequences finddirect use in methods for impacting pests, particularly insect pestssuch as pests of the order Homoptera. Accordingly, embodiments of theinvention provide new approaches for impacting insect pests that do notdepend on the use of traditional, synthetic chemical pesticides. Someembodiments involve the discovery of naturally-occurring, biodegradablepesticides and the genes that encode them.

Embodiments of the invention also encompass nucleic acid sequences thathave been optimized for expression by the cells of a particularorganism, for example nucleic acid sequences that have beenback-translated (i.e., reverse translated) using plant-preferred codonsbased on the amino acid sequence of a polypeptide having enhancedpesticidal activity. Further embodiments provide mutations which conferimproved or altered properties on polypeptides comprising them. Suchmutations may be utilized with any background sequence so long as theprovided toxin exhibits altered or improved pesticidal activity.

Embodiments of the present invention provide, inter alia, compositionsand methods for modulating the total level of polypeptides and/oraltering their ratios in a plant. As used herein, the term “modulation”is intended to mean an increase or decrease in a particular character,quality, substance, or response. The compositions comprise nucleotideand amino acid sequences from various plant species.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of embodiments of the present invention. Unless otherwisenoted, terms are to be understood according to conventional usage bythose of ordinary skill in the relevant art. Definitions of common termsin molecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5^(th) edition, Springer-Verlag; NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, “antimicrobial” or “antimicrobial activity” refers toantibacterial and antifungal activity, including, but not limited to theinhibition of pathogen growth.

As used herein, the terms “plant pathogen” or “plant pest” refer to anyorganism that can cause harm to a plant. A plant can be harmed by aninhibition or slowing of the growth of a plant, by damage to the tissuesof a plant, by a weakening of the immune system of a plant, by areduction in the resistance of a plant to abiotic stresses, by apremature death of the plant, and the like. Plant pathogens and plantpests include, but are not limited to insect pests of the orderHomoptera.

As used herein, the terms “disease resistance” or “pathogen resistance”are intended to mean that the organisms avoid the disease symptoms thatare the outcome of organism-pathogen interactions. That is, pathogensare prevented from causing diseases and the associated disease symptoms,or alternatively, the disease symptoms caused by the pathogen areminimized or lessened.

As used herein, the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured by butis not limited to pest mortality, pest weight loss, pest repellency, andother behavioral and physical changes of a pest after feeding andexposure for an appropriate length of time. In this manner, pesticidalactivity impacts at least one measurable parameter of pest fitness.Accordingly, “pesticidal activity” and “insecticidal activity” include,but are not limited to, damage caused by plant Homopteran insect pests.For example “pesticidal proteins” are proteins that display pesticidalactivity by themselves or in combination with other proteins. Endotoxinsare pesticidal proteins. Other examples of pesticidal proteins include,e.g., pentin-1 (see U.S. Pat. Nos. 6,057,491 and 6,339,144). A“pesticidal agent” will act similarly to suppress, control, and/or killan invading pathogen.

An “insecticidal composition” is intended to mean that the compositionsof embodiments of the invention have activity against plant insectpathogens; including insect pests of the order Homoptera, and thus arecapable of suppressing, controlling, and/or killing the invading insect.An insecticidal composition of the embodiments of the invention willreduce the symptoms resulting from insect challenge by at least about 5%to about 50%, at least about 10% to about 60%, at least about 30% toabout 70%, at least about 40% to about 80%, or at least about 50% toabout 90% or greater. Hence, the methods of the embodiments of theinvention can be utilized to protect organisms, particularly plants,from invading insects.

Assays that measure insecticidal activity are commonly known in the art,such as insect-feeding bioassays. See, for example, Marrone et al.(1985) J. Econ. Entomol. 78:290-293 and Czapla and Lang (1990) J. Econ.Entomol. 83:2480-2485, herein incorporated by reference. The preferreddevelopmental stage for testing for pesticidal activity is larvae orimmature forms of these above mentioned insect pests. The insects may bereared in total darkness at from about 20° C. to about 30° C. and fromabout 30% to about 70% relative humidity. Methods of rearing insectlarvae and performing bioassays are well known to one of ordinary skillin the art.

A wide variety of bioassay techniques are known to one skilled in theart. General procedures include addition of the experimental compound ororganism to the diet source in an enclosed container. Pesticidalactivity can be measured by, but is not limited to, changes inmortality, weight loss, attraction, repellency and other behavioral andphysical changes after feeding and exposure for an appropriate length oftime. Bioassays described herein can be used with any feeding insectpest in the larval or adult stage.

Compositions and methods for controlling Homopteran insect pests areprovided in the embodiments of the invention. The insecticidalcompositions comprise cyclotide nucleotide and amino acid sequences.Particularly, the plant nucleic acid and amino acid sequences andfragments and variants thereof set forth herein possess insecticidalactivity. Accordingly, the compositions and methods are useful inprotecting plants against Homopteran insect pests. Additionally providedare transformed plants, plant cells, plant tissues and seeds thereof.

The compositions of the embodiments of the invention can be used in avariety of methods whereby the protein products can be expressed in cropplants to function as insecticidal proteins. The compositions of theembodiments of the invention may be expressed in a crop plant such asmaize or soybean to function as an insecticidal agent. Expression of theproteins of the invention can also be altered, resulting in changes ormodulation of the level, tissue, or timing of expression in order toachieve enhanced insect resistance.

The coding sequence for the cyclotide can be used in combination with apromoter that is introduced into a crop plant. In one embodiment, ahigh-level expressing constitutive promoter may be utilized and wouldresult in high levels of expression of the cyclotide. In otherembodiments, the coding sequence may be operably linked to atissue-preferred promoter to direct the expression to a plant tissueknown to be susceptible to an insect. Likewise, manipulation of thetiming of expression may be utilized. For example, by judicious choiceof a promoter, expression can be enhanced early in plant growth to primethe plant to be responsive to insect attack. Likewise, pathogeninducible promoters can be used wherein expression of the cyclotide isturned on in the presence of the insect. If desired, a transit peptidecan be utilized to direct cellular localization of the protein product.In this manner, the native transit peptide or a heterologous transitpeptide can be used. However, it is recognized that both extracellularexpression and intracellular expression are encompassed by the methodsof the invention.

Sequences of the invention, as discussed in more detail below, encompasscoding sequences, antisense sequences, and fragments and variantsthereof. Expression of the sequences of the invention can be used tomodulate or regulate the expression of corresponding cyclotide proteins.

The compositions and methods of the invention can be used for enhancingresistance to plant Homopteran insect pests. The method involves stablytransforming a plant with a nucleotide sequence capable of modulatingthe plant insect defense system operably linked with a promoter capableof driving expression of a gene in a plant cell. By “enhancingresistance” increasing the tolerance of the plant to insects isintended. That is, the cyclotide may slow or prevent insect infectionand/or spread.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough, for example, antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-a-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic acid fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment that result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product. Eachof the proposed modifications is well within the routine skill in theart, as is determination of retention of biological activity of theencoded products.

Consequently, an isolated polynucleotide comprising a nucleotidesequence of at least 30 contiguous nucleotides derived from thenucleotide sequence of SEQ ID NOs: 1, 4, or 5 and its complement may beused in methods of selecting an isolated polynucleotide that affects theexpression of a plant cyclotide polypeptide in a host cell. For example,an isolated polynucleotide comprising at least 30, at least 40, at least50, at least 60 or at least any number of nucleotides up to the fulllength of SEQ ID NOs:1, 4 or 5. A method of selecting an isolatedpolynucleotide that affects the level of expression of a polypeptide ina virus or in a host cell (eukaryotic, such as plant or yeast,prokaryotic such as bacterial) may comprise the steps of: constructingan isolated polynucleotide or an isolated chimeric gene; introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;measuring the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of a polypeptide or enzyme activity in ahost cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms.

Specificity in hybridization is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of a complementary target sequence hybridizes to a perfectlymatched probe. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, severely stringent conditionscan utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower thanthe T_(m); moderately stringent conditions can utilize a hybridizationand/or wash at 6, 7, 8, 9, or 10° C. lower than the T_(m); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the T_(m).

Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). Also see Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl, 0.3 M trisodium citrate)at 50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. for at least 4 hours, often up to 12 hours or longer, and a finalwash in 0.1×SSC at 60 to 65° C. for at least 20 minutes, for example 30minutes, 40 minutes, 50 minutes, or 60 minutes. Optionally, wash buffersmay comprise about 1% SDS. Duration of hybridization is generally lessthan about 24 hours, usually about 4 to about 12 hours.

Thus, isolated sequences that encode a cyclotide polypeptide and whichhybridize under stringent conditions to the cyclotide sequencesdisclosed herein, or to fragments thereof, are encompassed byembodiments of the invention.

Substantially similar nucleic acid fragments of the invention may alsobe characterized by the percent identity of the amino acid sequencesthat they encode. For example, isolated nucleic acids which encode apolypeptide with a given percent sequence identity to the polypeptide ofSEQ ID NOs: 2, 3 or 6 are disclosed. Identity can be calculated using,for example, the BLAST, CLUSTALW or GAP algorithms under defaultconditions. The percentage of identity to a reference sequence is atleast 50% and, rounded upwards to the nearest integer, can be expressedas an integer selected from the group of integers consisting of from 50to 99. Thus, for example, the percentage of identity to a referencesequence can be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Examplesof such mathematical algorithms include, but are not limited to, thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thealignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264, as modified in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (Version 3.0,copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package of Genetics Computer Group, Version10 (available from Accelrys, 9685 Scranton Road, San Diego, Calif.,92121, USA). The scoring matrix used in Version 10 of the WisconsinGenetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915). Alignments using these programscan be performed using the default parameters. As used herein “defaultvalues” will mean any set of values or parameters which originally loadwith the software when first initialized.

The GAP program uses the algorithm of Needleman and Wunsch (1970) supra,to find the alignment of two complete sequences that maximizes thenumber of matches and minimizes the number of gaps. GAP considers allpossible alignments and gap positions and creates the alignment with thelargest number of matched bases and the fewest gaps. It allows for theprovision of a gap creation penalty and a gap extension penalty in unitsof matched bases. Default gap creation penalty values and gap extensionpenalty values in Version 10 of the Wisconsin Genetics Software Packagefor protein sequences are 8 and 2, respectively. For nucleotidesequences the default gap creation penalty is 50 while the default gapextension penalty is 3. The gap creation and gap extension penalties canbe expressed as an integer selected from the group of integersconsisting of from 0 to 200. Thus, for example, the gap creation and gapextension penalties can each be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater, up to and including200.

The CLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN and the ALIGN PLUS programs are based on the algorithm ofMyers and Miller (1988) supra.

The BLAST (Basic Local Alignment Search Tool) programs of Altschul etal. (1993) J. Mol. Biol. 215:403-410 are based on the algorithm ofKarlin and Altschul (1990) supra. BLAST nucleotide searches can beperformed with the BLASTN program, which searches a nucleotide queryagainst a nucleotide database, to obtain nucleotide sequences homologousto a nucleotide sequence encoding a protein of the invention. BLASTprotein searches can be performed with the BLASTX program, whichsearches a nucleotide query against a peptide database, to obtain aminoacid sequences homologous to a protein or polypeptide of the invention.The TBLASTN program provides for a peptide query against a nucleotidedatabase, while the TBLASTX program allows for a nucleotide queryagainst a nucleotide database with the translation of both to protein.To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules (see Altschul et al. (1997) supra). When utilizing anyBLAST program the default parameters of the respective programs can beused. Alignment may also be performed manually by inspection.

An “equivalent program” refers to any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by the preferred program.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e, gaps) compared to the reference sequence for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides in length, and optionally can be 30, 40,50, 100, or longer. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence, a gap penalty is typically introduced and issubtracted from the number of matches.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of a native(non-synthetic), endogenous sequence. A full-length polynucleotideencodes the full-length form of the specified protein.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, including at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitycompared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 60%, including at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, and 99%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C., depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, including 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequenceover a specified comparison window. Optimal alignment may be conductedusing the homology alignment algorithm of Needleman and Wunsch (1970)supra. An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as the BLAST programs discussed elsewhere inthis specification.(Altschul et al. (1993) supra).

Accordingly, a “substantial portion” of a nucleotide sequence comprisesa nucleotide sequence that will afford specific identification and/orisolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” a portion of the nucleotide sequence or a portion of theamino acid sequence, and hence protein, encoded thereby is intended. Thenucleic acid fragments of the instant invention may be used to isolatecDNAs and genes encoding homologous proteins from the same or otherplant species.

Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies. “PCR” or “polymerase chain reaction” isa technique used for the amplification of specific DNA segments (U.S.Pat. Nos: 4,683,195 and 4,800,159).

Genes encoding other plant cyclotides, either as cDNAs or genomic DNAs,could be isolated directly by using all or a portion of the instantnucleic acid fragments as DNA hybridization probes to screen librariesfrom any desired plant employing methodology well known to those skilledin the art. Specific oligonucleotide probes based upon the instantnucleic acid sequences can be designed and synthesized by methods knownin the art (Sambrook et al. (1989), supra). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 60 (or at least 40, or at least 30) contiguous nucleotides derivedfrom the nucleotide sequence set forth in SEQ ID NOs: 1, 4 or 5, and itscomplement, may be used in such methods to obtain a nucleic acidfragment encoding a substantial portion of an amino acid sequence of apolypeptide of the present invention.

Embodiments of the invention relate to a method of obtaining a nucleicacid fragment encoding a substantial portion of a cyclotide polypeptidecomprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least 10, at least 20, or atleast 30 or more contiguous nucleotides derived from the nucleotidesequence set forth in SEQ ID NOs:1, 4, or 5, and its complement; andamplifying a nucleic acid fragment using the oligonucleotide primer. Theamplified nucleic acid fragment preferably will encode a portion of aplant cyclotide polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the cyclotide amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Sambrook et al. (1989) supra).

Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence havecyclotide activity, for example, insecticidal activity, and therebyaffect responses to pathogens. Alternatively, fragments of a nucleotidesequence that are useful as hybridization probes generally do not encodeprotein fragments retaining biological activity. Thus, fragments of anucleotide sequence may range from at least about 20 nucleotides, about50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence encoding the proteins of the embodiments.

A fragment of a cyclotide nucleotide sequence that encodes abiologically active portion of a cyclotide protein of the invention willencode at least 10, 15, 25, 30, 50, 100, contiguous amino acids, or upto the total number of amino acids present in a full-length protein ofthe embodiments. Fragments of a cyclotide nucleotide sequence that areuseful as hybridization probes for PCR primers generally need not encodea biologically active portion of a cyclotide protein.

Thus, a fragment of a cyclotide nucleotide sequence may encode abiologically active portion of a cyclotide protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed herein. A biologically active portion of a cyclotideprotein can be prepared by isolating a portion of one of the cyclotidenucleotide sequences of the invention, expressing the encoded portion ofthe cyclotide protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the cyclotide protein.Nucleic acid molecules that are fragments of a cyclotide nucleotidesequence comprise at least 16, 20, 30, 40, 50, 75, 100, 150, 200, 250,or 300 nucleotides, or up to the number of nucleotides present in afull-length cyclotide nucleotide sequence disclosed herein.

By “variants” substantially similar sequences are intended. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the cyclotide polypeptides of the invention.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, such as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode acyclotide protein. Generally, variants of a particular nucleotidesequence of the invention will have at least about 50%, 60%, 65%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

By “variant protein” a protein derived from the native protein bydeletion (so-called truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein; deletionor addition of one or more amino acids at one or more sites in thenative protein; or substitution of one or more amino acids at one ormore sites in the native protein is intended. Variant proteinsencompassed by the embodiments are biologically active, that is theycontinue to possess the desired biological activity of the nativeprotein, that is, cyclotide activity as described herein, for example,insecticidal activity. Such variants may result from, for example,genetic polymorphism or from human manipulation. Biologically activevariants of a native cyclotide protein of the invention will have atleast about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, and including at least about 98%, 99% or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs described elsewhere hereinusing default parameters. A biologically active variant of the nativeprotein may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

The polypeptides of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Novel proteins having properties of interest may be createdby combining elements and fragments of proteins of the present inventionwith other proteins as well. Methods for such manipulations aregenerally known in the art. For example, amino acid sequence variants ofthe cyclotide proteins can be prepared by mutations in the DNA. Methodsfor mutagenesis and nucleotide sequence alterations are well known inthe art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (Macmillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include bothnaturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired cyclotide activity (for example, insecticidalactivity) or defense response activity. Obviously, mutations that willbe made in the DNA encoding the variant must not place the sequence outof reading frame and preferably will not create complementary regionsthat could produce secondary mRNA structure (see EP Patent PublicationNo. 0 075 444 B1).

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. Biological activity of thevariant polypeptides of the present invention can be assayed by anymethod known in the art, such as those already discussed and referencedelsewhere in this application.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different cyclotidecoding sequences can be manipulated to create a new cyclotide proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.Determination of preferred codons can be based on a survey of genesderived from the host cell where sequence information is available. Forexample, the codon frequency tables available on the world wide web atKazusa.or.jp/codon/may be used to determine preferred codons for avariety of organisms. See also Campbell and Gowri (1990) Plant Physiol.92:1-11; Murray et al. (1989) Nucleic Acids Res. 17:477-498, U.S. Pat.Nos. 5,380,831 and 5,436,391; and the information found on the worldwide web at agron.missouri.edu/mnl/77/10simmons.html; hereinincorporated by reference.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of thenucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell where sequence information is available.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct, including a nucleic acid expression cassettethat comprises a transgene of interest, the regeneration of a populationof plants resulting from the insertion of the transgene into the genomeof the plant, and selection of a particular plant characterized byinsertion into a particular genome location. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic makeup of a plant. The term “event” alsorefers to progeny produced by a sexual outcross between the transformantand another variety that include the heterologous DNA.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood to bewithin the scope of the invention and to comprise, for example, plantcells, protoplasts, tissues, callus, embryos, as well as flowers,ovules, stems, fruits, leaves, roots originating in transgenic plants ortheir progeny previously transformed with a DNA molecule of theinvention and therefore consisting at least in part of transgenic cells.

As used herein, the term “plant cell” includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. As used herein, the terms “encoding” or“encoded” when used in the context of a specified nucleic acid mean thatthe nucleic acid comprises the requisite information to guidetranslation of the nucleotide sequence into a specified protein. Theinformation by which a protein is encoded is specified by the use ofcodons. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA).

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequence mayconsist of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. While new promoters of various types useful in plant cellsare constantly being discovered; numerous examples of known promotersmay be found, for example, in the compilation by Okamuro and Goldberg(1989) Biochemistry of Plants 15:1-82. It is further recognized thatsince in most cases the exact boundaries of regulatory sequences havenot been completely defined, nucleic acid fragments of different lengthsmay have identical promoter activity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

Other methods known to enhance translation and/or mRNA stability canalso be utilized, for example, introns, such as the maize ubiquitinintron (Christensen and Quail (1996) Transgenic Res. 5:213-218 andChristensen et al. (1992) Plant Molecular Biology 18:675-689) or themaize Adhl intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48 andKyozuka et al. (1990) Maydica 35:353-357), and the like. Various intronsequences have been shown to enhance expression, particularly inmonocotyledonous cells. The introns of the maize AdhI gene have beenfound to significantly enhance the expression of the wild-type geneunder its cognate promoter when introduced into maize cells. Intron 1was found to be particularly effective and enhanced expression in fusionconstructs with the chloramphenicol acetyltransferase gene (Callis etal., (1987) Genes Develop. 1:1183-1200). In the same experimentalsystem, the intron from the maize bronzel gene had a similar effect inenhancing expression. The AdhI intron has also been shown to enhance CATexpression 12-fold (Mascarenhas et al. (1990) Plant Mol. Biol.6:913-920). Intron sequences have routinely been incorporated into planttransformation vectors, typically within the non-translated leader.

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

As used herein, “5′ leader sequence”, “translation leader sequence” or“5′ non-coding sequence” refer to that DNA sequence portion of a genebetween the promoter and coding sequence that is transcribed into RNAand is present in the fully processed mRNA upstream (5′) of thetranslation start codon. A 5′ non-translated leader sequence is usuallycharacterized as that portion of the mRNA molecule which most typicallyextends from the 5° CAP site to the AUG protein translation initiationcodon.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be an RNA sequencederived from post transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to a DNA that is complementaryto and derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense” RNA refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense”, when used in the context of a particularnucleotide sequence, refers to the complementary strand of the referencetranscription product. “Antisense RNA” refers to an RNA transcript thatis complementary to all or part of a target primary transcript or mRNAand that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous nucleotidesequence can be from a species different from that from which thenucleotide sequence was derived, or, if from the same species, thepromoter is not naturally found operably linked to the nucleotidesequence. A heterologous protein may originate from a foreign species,or, if from the same species, is substantially modified from itsoriginal form by deliberate human intervention.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Over expression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Under expression”refers to the production of a gene product in transgenic organisms atlevels below that of levels of production in normal or non-transformedorganisms. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Null mutant” refers here to a host cell that either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

In nature, some polypeptides are produced as complex precursors which,in addition to targeting labels such as the signal peptides discussedelsewhere in this application, also contain other fragments of peptideswhich are removed (processed) at some point during protein maturation,resulting in a mature form of the polypeptide that is different from theprimary translation product (aside from the removal of the signalpeptide). The following terms are of relevance. “Mature protein”,“preproprotein” or “prepropeptide” refer to a post-translationallyprocessed polypeptide; i.e., one from which any pre- or propeptidespresent in the primary translation product have been removed. “Precursorprotein” refers to the primary product of translation of mRNA; i.e.,with pre- and propeptides still present. Pre- and propeptides may be,but are not limited to, intracellular localization signals. The form ofthe translation product with only the signal peptide removed but notfurther processing yet is called a “propeptide” or “proprotein”. Thefragments to be removed may themselves are also referred to as“propeptides”. The skilled artisan will need to determine, depending onthe species in which the proteins are being expressed and the desiredintracellular location, if higher expression levels might be obtained byusing a gene construct encoding just the mature form of the protein, themature form with a signal peptide, or the proprotein (i.e., a formincluding propeptides) with a signal peptide. For optimal expression inplants or fungi, the pre- and propeptide sequences may be needed. Thepropeptides may play a role in aiding correct peptide folding.

A “chloroplast transit peptide” is an amino acid sequence that istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence that is translated in conjunction with aprotein and directs the protein to the secretory system (see Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal can furtherbe added, or if to the endoplasmic reticulum, an endoplasmic reticulumretention signal may be added. If the protein is to be directed to thenucleus, any signal peptide present should be removed and instead anuclear localization signal included (see Raikhel (1992) Plant Phys.100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below. Thus, isolated polynucleotides of the present inventioncan be incorporated into recombinant constructs, typically DNAconstructs, capable of introduction into and replication in a host cell.Such a construct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.,(1985; Supp. 1987) Cloning Vectors: A Laboratory Manual, Weissbach andWeissbach (1989) Methods for Plant Molecular Biology, (Academic Press,New York); and Flevin et al., (1990) Plant Molecular Biology Manual,(Kluwer Academic Publishers). Typically, plant expression vectorsinclude, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. Such plant expression vectors also can contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. (1989) supra.

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells that can be used to practice theinvention include, but are not limited to, yeast, bacterial, fungal,insect, amphibian, mammalian, and plant cells.

As used herein, “host cell” refers to a cell which comprises aheterologous nucleic acid sequence of the invention. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,fungal, insect, amphibian, mammalian or plant cells. Host plant cellsinclude monocotyledonous or dicotyledonous plant cells. One example of amonocotyledonous host cell is a maize host cell. One example of adicotyledonous host cell is a soybean host cell.

Over expression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

The cyclotide sequences of the invention are provided in expressioncassettes or DNA constructs for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa cyclotide sequence of the invention. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the cyclotide sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter),translational initiation region, a cyclotide polynucleotide sequence ofthe invention, a translational termination region and, optionally, atranscriptional termination region functional in the host organism. Thetranscriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. “Foreign” is intended to mean that the transcriptionalinitiation region is not found in the native plant into which thetranscriptional initiation region is introduced. As used herein, achimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould change expression levels of cyclotides in the host cell (e.g.,plant or plant cell). Thus, the phenotype of the host cell (e.g., plantor plant cell) is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase (NOS) termination regions. Seealso Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238); MDMVleader (Maize Dwarf Mosaic Virus), and human immunoglobulin heavy-chainbinding protein (BiP) (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Seealso, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones,and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724, and US Patent Publications US 20030083480A1 (now abandoned) and US 20040082770. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

In specific embodiments, methods for increasing pathogen resistance in aplant comprise stably transforming a plant with a DNA constructcomprising an antipathogenic nucleotide sequence of the inventionoperably linked to a promoter that drives expression in a plant. Suchmethods find use in agriculture particularly in limiting the impact ofplant pathogens on crop plants. While the choice of promoter will dependon the desired timing and location of expression of the anti-pathogenicnucleotide sequences, examples of promoters include constitutive andpathogen-inducible promoters.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in the host cell of interest. Suchconstitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);PEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, thosedisclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611, hereinincorporated by reference.

Generally, it will be beneficial to express the gene from an induciblepromoter, for example from a pathogen-inducible promoter. Such promotersinclude those from pathogenesis-related proteins (PR proteins), whichare induced following infection by a pathogen; e.g., PR proteins, SARproteins, beta-1, 3-glucanase, chitinase, etc. See, for example, Redolfiet al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992)Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116.See also WO 99/43819 published Sep. 9, 1999, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters. See, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156; herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced cyclotideexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. GenGenet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-specific promoters are known in the art. See, for example, Yamamotoet al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci.USA 90(20):9586-9590.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see WO 00/11177, hereinincorporated by reference). Gama-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

The method of transformation/transfection is not critical to the instantinvention. Various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be used with the instant invention.Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence to effect phenotypic changes in theorganism. The nucleic acid fragments of the instant invention may beused to create transgenic plants in which the disclosed plant cyclotidesare present at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of disease (e.g., fungal) andpathogen resistance in those cells. Thus, any method, which provides foreffective transformation/transfection may be employed.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; McCabe etal. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize);Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al.(1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.(1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, New York), pp.197-209(pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 andKaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediatedtransformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505(electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 andChristou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda etal. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacteriumtumefaciens); all of which are herein incorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensurethat expression of the desired phenotypic characteristic has beenachieved.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp., Pisum spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Hydrangea macrophylla), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In certain embodiments of the presentinvention, crop plants are used (for example, corn, soybean, alfalfa,sunflower, Brassica, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.).

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda derivedPL promoter and N-gene ribosome binding site (Simatake and Rosenberg(1981) Nature 292:128). Examples of selection markers for E. coliinclude, for example, genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235 and Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention. Such antimicrobial proteins can be used for anyapplication including coating surfaces to target microbes as describedfurther infra.

Synthesis of heterologous nucleotide sequences in yeast is well known.Sherman, et al. (1982) Methods in Yeast Genetics (Cold Spring HarborLaboratory) is a well recognized work describing the various methodsavailable to produce proteins in yeast. Two widely utilized yeasts forproduction of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like, as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques, radioimmunoassay,or other standard immunoassay techniques.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g. the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of proteins of the present invention areavailable, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo (1985)“Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector,” in DNA CloningVol. II: A Practical Approach, ed. D.M. Glover (IRL Press, Arlington,Va.), pp. 213-238.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler(1997) Biochemical Methods in Cell Culture and Virology (Dowden,Hutchinson and Ross, Inc.).

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,northern analysis of mRNA expression, western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum (ER) localization (Chrispeels (1991) supra), ornuclear localization signals (Raikhel (1992) supra) with or withoutremoving targeting sequences that are already present.

Unlike the promoter, which acts at the transcriptional level, suchtargeting information is part of the initial translation product. Thelocation of the protein in different compartments of the cell may makeit more efficacious or make it interfere less with the functions of thecell. For example, one may produce a protein preceded by a signalpeptide, which directs the translation product into the ER, by includingin the chimeric construct sequences encoding a signal peptide (suchsequences may also be called the “signal sequence”). The signal sequenceused could be that associated with the gene encoding the polypeptide, orit may be taken from another gene. There are many signal peptidesdescribed in the literature, and they are largely interchangeable(Raikhel N, Chrispeels M J (2000) Protein sorting and vesicle traffic.In B Buchanan, W Gruissem, R Jones, eds, Biochemistry and MolecularBiology of Plants. American Society of Plant Physiologists, Rockville,Md., pp 160-201, herein incorporated by reference). The addition of asignal peptide will result in the translation product entering the ER(in the process of which the signal peptide itself is removed from thepolypeptide), but the final intracellular location of the proteindepends on other factors, which may be manipulated to result inlocalization most appropriate for the pathogen and cell type.

The default pathway, that is, the pathway taken by the polypeptide if noother targeting labels are included, results in secretion of thepolypeptide across the cell membrane (Raikhel and Chrispeels, (2000)supra). This will leave the peptide between the cell membrane and cellwall, which will often be a suitable location. Other pathogens may bemore effectively combated by locating the peptide within the cell. Thiscan be accomplished, for example, by adding an ER retention signalencoding sequence to the sequence to the gene. Methods and sequences fordoing this are described in Raikhel and Chrispeels (2000) supra; forexample, adding sequences encoding the amino acids K, D, E and L in thatorder, or variations thereof described in the literature, to the end ofthe protein coding portion of the polypeptide will accomplish this.Alternatively, the use of vacuolar targeting labels such as thosedescribed by Raikhel and Chrispeels (2000) supra, in addition to asignal peptide will result in localization of the peptide in a vacuolarstructure. Use of a plastid transit peptide encoding sequence instead ofa signal peptide encoding sequence will result in localization of thepolypeptide in the plastid of the cell type chosen. One of skill in theart could also envision localizing the polypeptide in other cellularcompartments by addition of suitable targeting information. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of use may be discovered in thefuture.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one that allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

The instant polypeptides are useful in methods for impacting a plantpathogen comprising introducing into a plant or cell thereof at leastone nucleotide construct comprising a nucleotide sequence of theinvention operably linked to a promoter that drives expression of anoperably linked sequence in plant cells, wherein said nucleotidesequence is selected from the group consisting of: a nucleotide sequenceset forth in SEQ ID NOs: 1, 4 or 5; a nucleotide sequence that encodes apolypeptide having the amino acid sequence set forth in SEQ ID NOs: 2, 3or 6; a nucleotide sequence characterized by at least 85% sequenceidentity to the nucleotide sequences set forth in SEQ ID NOs: 1, 4 or 5;a nucleotide sequence characterized by at least 90% sequence identity tothe nucleotide sequence set forth in SEQ ID NOs: 1, 4 or 5; a nucleotidesequence characterized by at least 95% sequence identity to thenucleotide sequence set forth in SEQ ID NOs: 1, 4 or 5; and a nucleotidesequence that comprises the complement of any one of the above.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Polyclonal cyclotide antibodies can beprepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, orother mammal) with a cyclotide agent immunogen. The anti-cyclotideantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized antimicrobial polypeptides. At an appropriatetime after immunization, e.g., when the anti-cyclotide agent antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., New York); and Lerner (1981) Yale J. Biol. Med.54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-cyclotide antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a cyclotide to thereby isolateimmunoglobulin library members that bind the defensive agent. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening an antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734. The antibodies can be used to identifyhomologs of the cyclotides of the invention.

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and as markers for traitslinked to those genes. Such information may be useful in plant breedingin order to develop lines with desired phenotypes. For example, theinstant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Sambrook et al.(1989) supra) of restriction-digested plant genomic DNA may be probedwith the nucleic acid fragments of the instant invention. The resultingbanding patterns may then be subjected to genetic analyses usingcomputer programs such as MapMaker (Lander et al. (1987) Genomics1:174-181) in order to construct a genetic map. In addition, the nucleicacid fragments of the instant invention may be used to probe Southernblots containing restriction endonuclease-treated genomic DNAs of a setof individuals representing parent and progeny of a defined geneticcross. Segregation of the DNA polymorphisms is noted and used tocalculate the position of the instant nucleic acid sequence in thegenetic map previously obtained using this population (Botstein et al.(1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. in: Nonmammalian Genomic Analysis: APractical Guide, Academic Press, New York), 1996, pp. 319-346, andreferences cited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific I igation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

The methods of the invention can be used with other methods available inthe art for enhancing insect resistance in plants. For example,embodiments of the invention encompass any one of a variety of secondnucleotide sequences being utilized such that, when expressed in aplant, they help to increase the resistance of a plant to insect pests.It is recognized that such second nucleotide sequences may be used ineither the sense or antisense orientation depending on the desiredoutcome.

Furthermore, embodiments of the present invention may be effectiveagainst Homoptera such as aphids, planthoppers, leafhoppers, scaleinsects and others. The Homopteran order includes such families as theAdelgidae (adelgids), Aleyrodidae (whiteflies), Aphididae (aphids),Asterolecanidae (pit scales), Cercopidae (froghoppers or spittlebugs),Cicadellidae (leafhoppers), Cicadidae (cicadas), Cixiidae, Coccidae(soft scales), Dactylopiidae (dactylopiid or cochineal scales),Delphacidae (planthoppers), Diaspididae (armored scales), Eriococcidae(eriococcid scales), Flatidae (flatid planthoppers), Issidae (issidplanthoppers), Margarodidae (margarodid scales), Membracidae(treehoppers), Ortheziidae (ensign scales), Phoenicococcidae(phoenicococcid scales), Phylloxeridae (phylloxerans), Pseudococcidae(mealybugs) and Psyllidae (psyllids).

The methods of the invention can be used with other methods available inthe art for enhancing disease and pathogen resistance in plants.Similarly, the antimicrobial compositions described herein may be usedalone or in combination with other nucleotide sequences, polypeptides,or agents to protect against plant diseases and pathogens. Although anyone of a variety of second nucleotide sequences may be utilized,specific embodiments of the invention encompass those second nucleotidesequences that, when expressed in a plant, help to increase theresistance of a plant to pathogens.

Proteins, peptides, and lysozymes that naturally occur in insects(Jaynes et al. (1987) Bioassays 6:263-270), plants (Broekaert et al.(1997) Critical Reviews in Plant Sciences 16:297-323), animals (Vunnamet al. (1997) J. Peptide Res. 49:59-66), and humans (Mitra and Zang(1994) Plant Physiol. 106:977-981; Nakajima et al. (1997) Plant CellReports 16:674-679) are also a potential source of plant pathogenresistance (Ko, K. (2000) on the world wide web atScisoc.org/feature/BioTechnology/antimicrobial.html). Examples of suchplant resistance-conferring sequences include those encoding sunflowerrhoGTPase-Activating Protein (rhoGAP), lipoxygenase (LOX), AlcoholDehydrogenase (ADH), and Sclerotinia-Inducible Protein-1 (SCIP-1)described in U.S. patent application Ser. No. 09/714,767, hereinincorporated by reference. These nucleotide sequences enhance plantdisease resistance through the modulation of development, developmentalpathways, and the plant pathogen defense system. It is recognized thatsuch second nucleotide sequences may be used in either the sense orantisense orientation depending on the desired outcome.

In another embodiment, the cyclotides comprise isolated polypeptides ofthe invention. The cyclotides of the invention find use in thedecontamination of plant pathogens during the processing of grain foranimal or human food consumption; during the processing of feedstuffs,and during the processing of plant material for silage. In thisembodiment, the cyclotides of the invention are presented to grain,plant material for silage, or a contaminated food crop, or during anappropriate stage of the processing procedure, in amounts effective forantimicrobial activity. The compositions can be applied to theenvironment of a plant pathogen by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment, or dustingat a time when the plant pathogen has begun to appear or before theappearance of pests as a protective measure. It is recognized that anymeans that bring the cyclotide polypeptides in contact with the plantpathogen can be used in the practice of the invention.

Additionally, the compositions can be used in formulations used fortheir antimicrobial activities. Methods are provided for controllingplant pathogens comprising applying a decontaminating amount of apolypeptide or composition of the invention to the environment of theplant pathogen. The polypeptides of the invention can be formulated withan acceptable carrier into a composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, an emulsifiable concentrate, an aerosol, animpregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaricides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants, or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetmycotoxins. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the present invention are normally applied in theform of compositions and can be applied to the crop area or plant to betreated, simultaneously or in succession, with other compounds. In someembodiments, methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention (whichcontains at least one of the proteins of the present invention) arefoliar application, seed coating, and soil application.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7,9-tetraethyl-5-decyn-4, 7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate, oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate of a primary composition, whichrequires dilution with a suitable quantity of water or other diluentbefore application. The decontaminating concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly.

In a further embodiment, the compositions, as well as the polypeptidesof the present invention can be treated prior to formulation to prolongthe activity when applied to the environment of a plant pathogen as longas the pretreatment is not deleterious to the activity. Such treatmentcan be by chemical and/or physical means as long as the treatment doesnot deleteriously affect the properties of the composition(s). Examplesof chemical reagents include, but are not limited to, halogenatingagents; aldehydes such as formaldehyde and glutaraldehyde;anti-infectives, such as zephiran chloride; alcohols, such asisopropanol and ethanol; and histological fixatives, such as Bouin'sfixative and Helly's fixative (see, for example, Humason (1967) AnimalTissue Techniques (W.H. Freeman and Co.)).

In an embodiment of the invention, the compositions of the inventioncomprise a microbe having stably integrated the nucleotide sequence of acyclotide agent. The resulting microbes can be processed and used as amicrobial spray. Any suitable microorganism can be used for thispurpose. See, for example, Gaertner et al. (1993) in Advanced EngineeredPesticides, Kim (Ed.). In one embodiment, the nucleotide sequences ofthe invention are introduced into microorganisms that multiply on plants(epiphytes) to deliver the cyclotides to potential target crops.Epiphytes can be, for example, gram-positive or gram-negative bacteria.

It is further recognized that whole, i.e., unlysed, cells of thetransformed microorganism can be treated with reagents that prolong theactivity of the polypeptide produced in the microorganism when themicroorganism is applied to the environment of a target plant. Asecretion signal sequence may be used in combination with the gene ofinterest such that the resulting enzyme is secreted outside themicroorganism for presentation to the target plant.

In this manner, a gene encoding a cyclotide agent of the invention maybe introduced via a suitable vector into a microbial host, and saidtransformed host applied to the environment, plants, or animals.Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest may be selected for transformation. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, to provide for stable maintenance and expression of thegene expressing the detoxifying polypeptide, and for improved protectionof the proteins of the invention from environmental degradation andinactivation.

Such microorganisms include bacteria, algae, and fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Of particular interest are microorganisms, such as bacteria, e.g.,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., Saccharomyces, Pichia, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, Aureobasidium, andGliocladium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, Clavibacter xyli, and Azotobacter vinlandii; and phytosphereyeast species such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

The cyclotides of the invention can be used for any applicationincluding coating surfaces to target microbes. In this manner, targetmicrobes include human pathogens or microorganisms. Surfaces that mightbe coated with the cyclotides of the invention include carpets andsterile medical facilities. Polymer bound polypeptides of the inventionmay be used to coat surfaces. Methods for incorporating compositionswith antimicrobial properties into polymers are known in the art. SeeU.S. Pat. No. 5,847,047 herein incorporated by reference.

The present invention is further defined in the following Examples. Itshould be understood that these Examples are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, various modifications of theinvention, in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

EXAMPLES Example 1 Extraction and Isolation of Plant Cyclotides

Tissue from Viola spp. (10.0 g, wet weight) was harvested from plantsgrown in a growth chamber under standard conditions. The Viola spp.tissue was ground and extracted with buffer (50 mM Na₂HPO₄, 50 mMNaH₂PO₄, 50 mM Tris-HCl, 100 mM KCl, 2 mM EDTA). The crude extract wasfiltered through a cotton-plug filter to remove plant debris while fineparticulate matter was removed by centrifugation (Sorvall® InstrumentsRC5C, 15,000 rpm, 15 minutes, 4° C.). The supernatant was partitionedwith n-Butanol (BuOH). The BuOH layer was dried in a speedvac andredissolved in 2 mL distilled water. The sample (100 μL/run) wasfractionated by reverse phase high performance liquid chromatography,RP-HPLC.

Example 2 Fractionation by RP-HPLC

RP-HPLC was performed on a Hewlett-Packard HP1100 series using a Vydac®300 angstrom pore size, 10 microns particle C18 column (catalog number218TP104, Grace Vydac, W.R. Grace & Co., Columbia, Md.) and a 0-80%gradient from Solvent A (95% H₂O, 5% acetonitrile, 0.1% trifluoroaceticacid) to Solvent B (5% H₂O, 95% acetonitrile, 0.1% trifluoroacetic acid)over 40 minutes with a flow rate of 0.6 mL/min. Samples for bioassayagainst the target homopteran pests were collected in 96-well plates ona Foxy™ 200 fraction collector (Isco, Inc., Lincoln, Nebr.). The plateswere lyophilized and assayed against different targets in replicates ofthree. Peptides having specific activity against Peregrinus maidis andAphis glycines were purified to homogeneity on a capillary reverse phaseC18 column (Magic 2002 HPLC System, Michrom BioResources, FUTECS, DaejonKorea) utilizing the following gradient: 10-30% Solvent B over 10minutes followed by 30-60% Solvent B over 60 minutes; and subsequentlyassayed in a dose-dependent or dose-response manner.

The HPLC profile of the crude extract of Viola spp. is shown in FIG. 1.The eluted peak corresponding to the cyclotide of the instant inventionis labeled E5, with an observed mass of 3158.3 Da.

Example 3 Mass Spectrometry

Mass spectra were acquired on a Micromass Platform LCZ instrument(Waters Mass Spectrometry Systems, Micromass Division, Manchester, U.K.)during LCMS runs. Dried samples of the crude extract were dissolved indistilled water to give a concentration of approximately 1 mg/mL, and 10μL was injected into the solvent stream for introduction into theionization source of the mass spectrometer. Mass spectra were obtainedover the range 900-2200 m/z⁺ and processed using the software MassLynx™,version 3.1. The gradient was started at 0% buffer B (5% H₂O, 95%acetonitrile, 0.1% trifluoroacetic acid) to 75% in 30 minutes with aflow rate of 50 μL. The mass of the cyclotide E5 which was obtained fromthe mass spectrometry procedure was 3158.3 Da.

Example 4 Production of Viola spp. cDNA Libraries

Total RNA from Viola spp. leaves was prepared by pulverizing the tissuewith a mortar and pestle in liquid nitrogen and lysing cells in thepresence of TRIzol™ (Invitrogen Life Technologies, Carlsbad, Calif.)according to the manufacturer's protocol. Viola leaves were harvesteddirectly into liquid nitrogen before processing. PolyA(+) RNA wasoligo(dT)-cellulose affinity column purified from total RNA using themRNA Purification Kit (Amersham Pharmacia Biotech, CA) and following thekit's protocol in preparation for cDNA library construction. Firststrand cDNA synthesis was performed using Superscript II™ (InvitrogenLife Technologies) and subsequent second strand synthesis, linkeraddition, and directional cloning into the EcoRI and XhoI sites ofpBlueScript™SK+(Stratagene, La Jolla, Calif.) was performed according tothe instructions provided with the Stratagene cDNA kit (Stratagene).cDNA was purified using a cDNA column (Invitrogen Life Technologies)immediately prior to ligation into the vector.

Sequencing of cDNA library clones was performed using the ABI PRISM™BigDye Terminator Cycle Sequencing Ready reaction kit with FS AmpliTaq™DNApolymerase (Applied Biosystems, Foster City, Calif.) and analyzed on anABI Model 373 Automated DNA Sequencer (Applied Biosystems).

Example 5 N-terminal Sequencing

Approximately 1.0 μg of cyclotide E5 was reduced with TCEP and alkylatedwith maleimide. It was subsequently cleaved with Endo-Glu C to yield alinear chain peptide. The mass of the peptide was monitored at eachstage. The N-terminal tag of the cleaved species was sequenced using anautomatic Edman sequencer (494 Protein Sequencer, Applied Biosystems,Foster City, Calif.) for 31 cycles. Peptide sequences corresponding tothose obtained by amino acid sequencing of the Endo GluC treated activeswere used to compare to the corresponding cDNA clone sequence librarytranslated in all 6 reading frames using TBLASTN or TFASTA programs.Sequences having 100% identity to the experimentally generated aminoacid sequence(s) were fully translated and their predicted molecularweight (MW) compared to the MW of purified active protein. In this way asingle nucleotide sequence that encodes the exact amino acid sequence ofcyclotide E5 with a theoretical mass (3159.84 Da) that closely matchedthe experimentally observed mass (3158.3 Da) within the error limit wasidentified as the nucleotide corresponding to the peptide of thisinvention.

The following complete amino acid sequence was obtained: N-terminalsequence (SEQ ID NO:3): KIPCGESCVYIGCTLTALAGCKCKNKVCYN.

Example 6 Insecticidal Activity of Cyclotide E5 Tested AgainstHomopteran Insects Peregrinus maidis and Aphis glycines

To test for the insecticidal activity of cyclotide E5 against the targethomopteran pests, the lyophilized fractionated samples were placed in96-well microtiter plates, resuspended in H₂O and added to a 20% sucrosesolution as detailed below. Parafilm was stretched to just short of itsbreaking point and used to seal the sucrose/sample reservoir. Adultinsects were then added on top of the membrane in a second chamber suchthat the base of the insect enclosure was the parafilm membrane. The topof the insect chamber was then sealed with plastic wrap and puncturedfor air exchange. The number of insects feeding from each well wasdetermined. The assay was scored after 72 hours and nsecticidal activitywas measured based on insect mortality.

Specifically, for the dose-response assay results shown in Tables 1 and2, lyophilized RP-HPLC-pure sample was resuspended in 300 pL H₂O andadded to 20% sucrose in the following volumes: 60, 45, 25, 10, and 5 μL.Two replicates per dose were prepared, including two negative controls.After 72 hours, the wells were scored for insect mortality. Thefollowing results were observed. TABLE 1 Dose response assay for E5against Peregrinus maidis E5 volume (μL) 60 45 25 10 5 0 0 E5Concentration (μM) 33.22 16.61 9.97 4.09 2.73 0 0 average percent (%)100 100 95 90 90 0 0 insect mortality

TABLE 2 Dose response assay for E5 against Aphis glycines E5 Volume (μL)60 45 25 10 5 0 0 E5 Concentration (μM) 76.35 57.26 31.81 12.73 6.36 0 0average percent (%) 100 95 75 80 56.5 10.5 4.2 insect mortality

1. An isolated cyclotide comprising the amino acid sequence set forth inSEQ ID NO: 2, 3 or
 6. 2. The polypeptide of claim 1, wherein saidpolypeptide is characterized by insecticidal activity against at leastone plant Homopteran pest.
 3. The polypeptide of claim 2, wherein saidplant Homopteran pest is Peregrinus maidis.
 4. The polypeptide of claim2, wherein said plant Homopteran pest is Aphis glycines.